Methods and Systems of Multi-Assay Processing and Analysis

ABSTRACT

The instant disclosure provides methods of multi-assay processing and multi-assay analysis. Such multi-assay processing and analysis pertain to automated detection of target nucleic acids, e.g., as performed in the clinical setting for diagnostic purposes. Also provided are common assay timing protocols derived from a variety of individual nucleic acid amplification and analysis protocols and modified to prevent resource contention. The instant disclosure also provides systems and devices for practicing the methods as described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. § 119(e), this application claims priority to thefiling date of the U.S. Provisional Patent Application Ser. No.62/308,625, filed Mar. 15, 2016, the disclosure of which application isherein incorporated by reference.

BACKGROUND

Molecular diagnostic assays, including nucleic acid amplification basedmethods, have become a mainstay of clinical medicine and the variety ofavailable tests and the demand for such tests by clinicians hasincreased dramatically. This demand places increasing pressures onclinical laboratories to process, not only a greater volume of samples,but also a greater diversity of tests on the samples. Thus, there is aburden on clinical testing facilities to efficiently perform a widerrange of different nucleic acid amplification based tests.

Assay protocols define all the reagents, processing steps, processingtimes, temperature profiles, etc., required to process a sample throughan automated instrument in order to obtain a diagnostic result.Historically, unique assay protocols containing varying reagents, stepsand times are developed for each type of assay in order to optimizeassay performance. For instruments that process samples in batch mode,where only one type of assay is processed per run, having unique assayprotocols does not impact the overall system throughput and schedulingcomplexity since the protocol is the same for all samples being run inthe batch. However, in the case of instruments that process multipleassay types simultaneously per run, unique assay protocols have asignificant impact on the scheduler complexity and efficient use ofsystem resources.

SUMMARY

Aspects of the instant disclosure include methods for multi-assayprocessing and multi-assay quantification and multi-assay processingsystems.

Aspects of the instant disclosure include a method of multi-assayprocessing that includes: a) preparing a sample processing unit (SPU)cartridge for each of two or more different target nucleic aciddetection assays; b) loading a sample into each prepared SPU cartridge;c) processing each loaded SPU cartridge to isolate a sample nucleic acidfor each of the two or more different target nucleic acid detectionassays; and d) amplifying and analyzing each sample nucleic acid for atarget nucleic acid specific to each of the two or more different targetnucleic acid detection assays, wherein the method comprises at least onedelay step within or between steps a) through d) and steps a) through d)are each performed for a time period that is equal for the two or moredifferent target nucleic acid detection assays. In some instances,aspects of the method include a delay step between steps a) and b), adelay step between steps b) and c) and/or a delay step between steps c)and d).

In some instances, aspects of the method include rehydrating lyophilizedreagents for each of the two or more different target nucleic aciddetection assays prior to the preparing, wherein the rehydrating isperformed for a time period that is equal for the two or more differenttarget nucleic acid detection assays. In some instances, aspects of themethod include a delay step following the rehydrating.

In some instances, aspects of the method include pre-treating eachloaded SPU cartridge prior to the processing, wherein the pre-treatingis performed for a time period that is equal for the two or moredifferent target nucleic acid detection assays. In some instances,aspects of the method include a delay step following the pre-treating.

In some instances, aspects of the method include where the pre-treatingcomprises contacting the sample with a protease.

In some instances, aspects of the method include where the processingcomprises transferring the sample into a solution comprising a lysisbuffer, wherein the transferring is performed for a time period that isequal for the two or more different target nucleic acid detectionassays. In some instances, aspects of the method include a delay stepfollowing the transferring.

In some instances, aspects of the method include where the processingcomprises eluting the nucleic acid and transferring the eluted nucleicacid into a reaction vessel for the amplifying and analyzing, whereinthe eluting is performed for a time period that is equal for the two ormore different target nucleic acid detection assays. In some instances,aspects of the method include a delay step following the eluting.

In some instances, aspects of the method include where two or moredifferent target nucleic acid detection assays include an assay todetect a human immunodeficiency virus (HIV) nucleic acid, an assay todetect a hepatitis C virus (HCV) nucleic acid, an assay to detect ahepatitis B virus (HBV) nucleic acid, an assay to detect a Chlamydiatrachomatis (CT) nucleic acid, a Neisseria gonorrhoeae (NG) nucleic acidor a combination there of, an assay to detect a Human papillomavirus(HPV) nucleic acid, an assay to detect a Cytomegalovirus (CMV) nucleicacid, an assay to detect an Epstein-Barr virus (EBV) nucleic acid, anassay to detect a BK virus nucleic acid, an assay to detect aMethicillin-resistant Staphylococcus aureus (MRSA) nucleic acid, anassay to detect a Clostridium difficile (D. Diff.) nucleic acid, anassay to detect a Vancomycin-resistant Enterococcus (VRE) nucleic acid,an assay to detect an Adenovirus nucleic acid, an assay to detect atuberculosis (TB) nucleic acid, an assay to detect a Varicella-zostervirus (VZV) nucleic acid, an assay to detect a Herpes simplex virus(HSV) nucleic acid, an assay to detect a JC virus nucleic acid, an assayto detect an Enterovirus nucleic acid, an assay to detect aLymphogranuloma venereum (LGV) nucleic acid, an assay to detect aRespiratory Viral Panel (RVP) nucleic acid, an assay to detect a humanherpesvirus 6 (HHV6) nucleic acid, an assay to detect a Trichomonas(Trich) nucleic acid, a Mycoplasma (Myco) nucleic acid or a combinationthereof, and/or an assay to detect a Norovirus nucleic acid. In someinstances, aspects of the method include processing 3 or more differenttarget nucleic acid detection assays. In some instances, aspects of themethod include processing 10 or more different target nucleic aciddetection assays.

Aspects of the instant disclosure include a method of multi-assayquantification that includes: a) initiating a nucleic acid amplificationprotocol in a first sample pair; b) scanning the first sample pair withan optical detector at a regular interval during the nucleic acidamplification protocol, wherein the interval allows for the collectionof data by the optical detector at timepoints of the amplificationprotocol sufficient for quantification of the nucleic acid amplificationin the first sample pair; c) initiating the nucleic acid amplificationprotocol in a second sample pair at a time that allows the second samplepair to be scanned by the optical detector at the regular intervals andcollection of data by the optical detector at timepoints of theamplification protocol sufficient for quantification of nucleic acidamplification in the second sample pair.

In some instances, aspects of the multi-assay quantification methodinclude initiating the nucleic acid amplification protocol of the firstsample pair and initiating the nucleic acid amplification protocol ofthe second sample pair at essentially the same time. In some instances,aspects of the multi-assay quantification method include initiating thenucleic acid amplification protocol of the first sample pair andinitiating the nucleic acid amplification protocol of the second samplepair at different times. In some instances, aspects of the multi-assayquantification method include where the scanning is performed three ormore times during the nucleic acid amplification protocol. In someinstances, aspects of the multi-assay quantification method includewhere the interval allows for the collection of data by the opticaldetector at more timepoints of the amplification protocol than necessaryfor quantification of the nucleic acid amplification in the first andsecond sample pairs.

In some instances, aspects of the multi-assay quantification methodinclude initiating the nucleic acid amplification protocol in a thirdsample pair at a time that allows the third pair to be scanned by theoptical detector at the regular intervals and collection of data by theoptical detector at timepoints of the amplification protocol sufficientfor quantification of nucleic acid amplification in the third samplepair. In some instances, aspects of the multi-assay quantificationmethod include initiating the nucleic acid amplification protocol of thefirst, second and third sample pairs at essentially the same time. Insome instances, aspects of the multi-assay quantification method includeinitiating of the nucleic acid amplification protocol of the first,second and third sample pairs at different times.

In some instances, aspects of the multi-assay quantification methodinclude initiating the nucleic acid amplification protocol in a fourthsample pair at a time that allows the fourth pair to be scanned by theoptical detector at the regular intervals and collection of data by theoptical detector at timepoints of the amplification protocol sufficientfor quantification of nucleic acid amplification in the fourth samplepair. In some instances, aspects of the multi-assay quantificationmethod include initiating of the nucleic acid amplification protocol ofthe first, second, third and fourth sample pairs at essentially the sametime. In some instances, aspects of the multi-assay quantificationmethod include initiating of the nucleic acid amplification protocol ofthe first, second, third and fourth sample pairs at different times.

In some instances, aspects of the multi-assay quantification methodinclude initiating the nucleic acid amplification protocol in a fifthsample pair at a time that allows the fifth pair to be scanned by theoptical detector at the regular intervals and collection of data by theoptical detector at timepoints of the amplification protocol sufficientfor quantification of nucleic acid amplification in the fifth samplepair. In some instances, aspects of the multi-assay quantificationmethod include initiating of the nucleic acid amplification protocol ofthe first, second, third, fourth and fifth sample pairs at essentiallythe same time. In some instances, aspects of the multi-assayquantification method include initiating of the nucleic acidamplification protocol of the first, second, third, fourth and fifthsample pairs at different times.

In some instances, aspects of the multi-assay quantification methodinclude initiating the nucleic acid amplification protocol in a sixthsample pair at a time that allows the sixth pair to be scanned by theoptical detector at the regular intervals and collection of data by theoptical detector at timepoints of the amplification protocol sufficientfor quantification of nucleic acid amplification in the sixth samplepair. In some instances, aspects of the multi-assay quantificationmethod include initiating of the nucleic acid amplification protocol ofthe first, second, third, fourth, fifth and sixth sample pairs atessentially the same time. In some instances, aspects of the multi-assayquantification method include the initiating of the nucleic acidamplification protocol of the first, second, third, fourth, fifth andsixth sample pairs at different times.

Aspects of the instant disclosure include a multi-assay processingsystem including: a) a sample processing unit (SPU) cartridgepreparation module; b) a sample loading module; c) a SPU processingmodule; d) a nucleic acid amplification and analysis module; and e)control circuitry configured to perform a method as herein described.

In some instances, aspects of the system include a module forrehydrating lyophilized reagents. In some instances, aspects of thesystem include an SPU processing module configured for pre-treating eachsample prior to processing the sample. In some instances, aspects of thesystem include a reaction transfer module. In some instances, aspects ofthe system include a single robotic pipette resource that functions inthe SPU cartridge preparation module. In some instances, aspects of thesystem include where the single robotic pipette resource also functionsin the sample loading module, the module for rehydrating lyophilizedreagents and/or the reaction transfer module. In some instances, aspectsof the system include one or more bulk filling robots. In someinstances, aspects of the system include a single bulk filling robot.

In some instances, aspects of the system include one or more wasterobots. In some instances, aspects of the system include a single wasterobot. In some instances, aspects of the system include one or more SPUcartridge handling robots. In some instances, aspects of the systeminclude a single SPU cartridge handling robot.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides an idealized lockstep protocol used to harmonize theprocessing of samples for different nucleic acid assays related to HIV,HCV, CT/NG and HBV diagnostic protocols.

FIG. 2 demonstrates how four different assay types (HIV, HCV, CT/NG andHBV), utilizing a common lockstep assay timing protocol, can beprocessed over three runs given an idealized system with essentiallylimitless resources. Resource contention is indicated.

FIG. 3 demonstrates a sequence with appropriately placed timing gaps(i.e., delays) resulting in a fixed cadence of sample input that allowsfor a single lockstep timing protocol in a system with finite resources.

FIG. 4 demonstrates how three different assays, each with four samples,can be configured during amplification and detection for coordinatedmeasurements according to an embodiment of the disclosure.

FIG. 5 depicts a flow chart for a conventional sample processingprotocol performed in an automated system subject to resourcecontention.

FIG. 6 depicts a flow chart for an embodiment of the present methodsemploying a lockstep protocol with scheduled delays between processsteps.

DEFINITIONS

The term “analyte” as used herein an analyte refers to a target moleculeto be detected in a sample wherein detection of the analyte may beindicative of a biological state of the organism from which the samplewas derived. For example, where an analyte is a nucleic acid analyte,detection of the nucleic acid analyte may be indicative of a biologicalstate of the organisms from which the sample was derived including e.g.,where detection of viral nucleic acid may indicate infection with aparticular pathogen, etc.

The term “reaction vessel” as used herein generally referrers to acontainer within which an amplification reaction is performed. Suchreaction vessels may be obtained from commercial sources, e.g., asoff-the-shelf components, or may be custom manufactured. Reactionvessels useful in nucleic acid amplification reactions will generally becapable of rapidly transferring heat across the vessel, e.g., throughthe use of highly conductive materials (e.g., thermally conductiveplastics) or physical modifications of the vessel (e.g., thin walls).Common reaction vessels include but are not limited to e.g., tubes,vials, multi-well plates, and the like. Reaction vessels may beconstructed of a variety of materials including but not limited to e.g.,polymeric materials. In some instances, a method as described herein maybe configured for use with a reaction vessel and/or reaction vesselsystem as described in e.g., Attorney Docket No. ADDV-056WO, whichclaims priority to U.S. Ser. No. 62/308,620, the disclosures of whichare incorporated herein by reference in their entireties.

The term “assessing” includes any form of measurement, and includesdetermining if an element is present or not. The terms “determining”,“measuring”, “evaluating”, “assessing” and “assaying” are usedinterchangeably and include quantitative and qualitative determinations.Assessing may be relative or absolute. “Assessing the identity of”includes determining the most likely identity of a particular compoundor formulation or substance, and/or determining whether a predictedcompound or formulation or substance is present or absent. “Assessingthe quality of” includes making a qualitative or quantitative assessmentof quality e.g., through the comparisons of a determined value to areference or standard of known quality.

The term “bodily fluid” as used herein generally refers to fluidsderived from a “biological sample” which encompasses a variety of sampletypes obtained from an individual or a population of individuals and canbe used in a diagnostic, monitoring or screening assay. The definitionencompasses blood and other liquid samples of biological origin. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by mixing or pooling of individualsamples, treatment with reagents, solubilization, or enrichment forcertain components, such as nucleated cells, non-nucleated cells,pathogens, etc.

The term “biological sample” encompasses a clinical sample, and alsoincludes cells in culture, cell supernatants, cell lysates, serum,plasma, biological fluid, and tissue samples. The term “biologicalsample” includes urine, saliva, cerebrospinal fluid, interstitial fluid,ocular fluid, synovial fluid, blood fractions such as plasma and serum,and the like.

The terms “control”, “control assay”, “control sample” and the like,refer to a sample, test, or other portion of an experimental ordiagnostic procedure or experimental design for which an expected resultis known with high certainty, e.g., in order to indicate whether theresults obtained from associated experimental samples are reliable,indicate to what degree of confidence associated experimental resultsindicate a true result, and/or to allow for the calibration ofexperimental results. For example, in some instances, a control may be a“negative control” assay such that an essential component of the assayis excluded such that an experimenter may have high certainty that thenegative control assay will not produce a positive result. In someinstances, a control may be “positive control” such that all componentsof a particular assay are characterized and known, when combined, toproduce a particular result in the assay being performed such that anexperimenter may have high certainty that the positive control assaywill not produce a positive result. Controls may also include “blank”samples, “standard” samples (e.g., “gold standard” samples), validatedsamples, etc.

By “control circuitry” or “data processing unit”, as used herein, ismeant any hardware and/or software combination that will perform thefunctions required of it. For example, any data processing unit hereinmay be a programmable digital microprocessor such as available in theform of an electronic controller, mainframe, server or personal computer(desktop or portable). Where the data processing unit is programmable,suitable programming can be communicated from a remote location to thedata processing unit, or previously saved in a computer program product(such as a portable or fixed computer readable storage medium, whethermagnetic, optical or solid state device based). In some instances,control circuitry or a data processing unit of the present disclosuremay be specifically programmed to perform the functions required of itand may thus be referred to as a special purpose computer.

By “lockstep” or “lockstep protocol” is meant a protocol where the stepsof the protocol follow one another as closely as possible. In someinstances described herein a lockstep protocol may be determined basedon corresponding steps of different protocols where such protocols willbe performed in parallel or concomitantly. Thus, a lockstep protocolneed not consist of only successive shortest steps of a particularprotocol but may instead include one or more longest steps of variousprotocols that are to be performed in parallel.

By “cadence” is meant batch per unit time and, as it relates to alockstep protocol, a cadence may relate to a regular or fixed point ortime of sample input or sample processing initiation. Accordingly, aregular cadence may refer to the initiation of a batch at regular timeintervals.

By “resource contention”, as used herein, is meant a conflict overaccess to a shared resource of an integrated system. Resource contentionmay apply to the physical components of a system where such componentsare limiting to progression of a process. For example, where two modulesof a system utilize a shared resource such resource may be in contentionif/when both modules require the resource simultaneously.

By “batch”, as used herein, is meant a grouping of common samples orassays processed in parallel according to a single protocol having acommon initiation time. Samples or assays within batches of the instantdisclosure may or may not be processed exactly alike but will generallyinitiate and terminate together. For example, samples and assays of thebatch may be processed exactly alike throughout the entire protocolincluding e.g., where sample preparation, processing andamplification/analysis are identical for all samples or assays of thebatch. In other instances, samples and assays of the batch may not beprocessed exactly alike throughout the entire protocol including e.g.,where one or more of sample preparation, processing and/oramplification/analysis are not identical for all samples or assays ofthe batch.

DETAILED DESCRIPTION

The instant disclosure provides methods of multi-assay processing andmulti-assay analysis. Such multi-assay processing and analysis pertainto automated detection of target nucleic acids, e.g., as performed inthe clinical setting for diagnostic purposes. Also provided are commonassay timing protocols derived from a variety of individual nucleic acidamplification and analysis protocols and modified to prevent resourcecontention. The instant disclosure also provides systems and devices forpracticing the methods as described herein.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating un-recited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Methods

The instant disclosure provides methods of multi-assay processing whereby “multi-assay” is meant multiple, two or more, different assays.Multi-assay processing and/or analysis may be performed by a singlemolecular analysis device including those molecular analysis deviceshaving limited physical resources. In many embodiments, the instantmethods pertain to multi-assay processing of nucleic acid amplificationand analysis assays including but not limited to e.g., those involvingPCR methods, including e.g., real-time PCR methods.

The PCR process is a nucleic acid amplification method whereby a targetnucleic acid sequence is amplified by a factor of 2^(n) by repeating (1)a denaturing temperature (e.g., of 95° C.) that serves to denature thetwo strands of a double stranded nucleic acid template; (2) an annealingtemperature (e.g., on the order of 55° C. to 65° C.) that serves toanneal one or more complementary nucleic acids to a single strand of thedenatured nucleic acid; and (3) an extension temperature that providesthe permissive temperature for a nucleic acid polymerase to extend thecomplementary nucleic acid according to the sequence of the template,alternately n times (referred as a “thermal cycle”).

In real-time PCR, the amount of nucleic acid is measured at a pluralityof time points during the amplification reaction to determine the actualor relative amount of target nucleic acid analyte initially present inthe sample. Real-time PCR may be quantitative, semi-quantitative orqualitative. Real-time PCR is generally carried out in a thermal cyclerwith the capacity to illuminate each amplification sample with a beam oflight of at least one specified wavelength and detect the fluorescenceemitted by an excited fluorophore that is either incorporated into theamplicon or unquenched during amplification. Non-specific fluorochromes(e.g., DNA binding dyes such as e.g., SYBR Green) or specificfluorescent hybridization probes may be used. Using different-coloredlabels, fluorescent probes can be used in multiplex assays formonitoring several target sequences in the same tube.

One method of using fluorescently labeled probes relies on a DNA-basedprobe with a fluorescent reporter at one end and a quencher offluorescence at the opposite end of the probe. The close proximity ofthe reporter to the quencher prevents detection of its fluorescence.When bound to a target sequence, breakdown of the probe by the 5′ to 3′exonuclease activity of the polymerase breaks the reporter-quencherproximity and thus allows unquenched emission of fluorescence, which canbe detected after excitation with a particular wavelength of light. Anincrease in the product targeted by the reporter probe at each PCR cycletherefore causes a proportional increase in fluorescence due to thebreakdown of the probe and release of the reporter. Any convenientpolymerase with 5′ to 3′ exonuclease activity may find use in suchassays including but not limited to wild-type Taq polymerase andmodified or engineered polymerases including but not limited to e.g.,those available from commercial suppliers such as e.g., New EnglandBiolabs (Ipswich, Mass.), Life Technologies (Carlsbad, Calif.), SigmaAldrich (St. Louis, Mo.) and Kapa Biosystems, Inc. (Wilmington, Mass.)such as e.g., KAPA2G DNA Polymerases.

Various real-time PCR assays find use in clinical diagnostics includingdetection of a target nucleic acid of an infectious agent. As usedherein an “infectious agent” includes any biological pathogen that mayinfect a host, where such pathogens have a nucleic acid component, e.g.,a nucleic acid genome, that may be detected, referred to herein as a“target nucleic acid” in an assay as described herein. As suchinfectious agents of the instant disclosure will vary and may includebut are not limited to e.g., parasites, bacteria, yeast, fungi, viruses,and the like. The instant methods and systems may be applied to anyinfectious agent having a nucleic acid component that may be detected byPCR methods, including real-time PCR and reverse transcription (RT) PCR,including real-time RT-PCR. As such, a target nucleic acid of aninfectious agent may be DNA or RNA, including but not limited to e.g.,single stranded DNA, double stranded DNA, single stranded RNA, doublestranded RNA, and the like.

Multi-assay methods and systems find use in automated detection oftarget nucleic acids for a plurality of different assays, includingmultiple different clinically relevant nucleic acid detection assays. Insome instances, multi-assay methods may apply multiple different assaysto a single biological sample including e.g., where a single sample isdivided into aliquots and each aliquot is applied to two or moredifferent nucleic acid detection assays. In some instances, multi-assaymethods may apply multiple different assays to different biologicalsamples including e.g., where different biological samples may bederived from different tissues of a single subject, derived from thesubject at different times, derived from different subjects, etc.

In some instances, the methods and systems as described herein involvesimultaneous or co-timely or overlapping detection of a plurality oftarget nucleic acids derived from an organism. Organisms from whichtarget nucleic acids may be derived include clinically relevant andnon-clinically relevant organisms. Non-clinically relevant organisms mayinclude e.g., organisms useful in research applications, organismsuseful in industrial applications, organisms useful in agriculturalapplications, organisms of environmental concern, etc.

In some instances, a multi-assay processing, analysis or detectionmethod may find use in simultaneous or co-timely or overlappingprocessing, analysis or detection of a plurality of clinically relevanttarget nucleic acids including but not limited to e.g., target nucleicacids derived or originating from one or more clinically relevantpathogens such as e.g., Acinetobacter baumannii, Acinetobacter lwoffii,Acinetobacter spp. (incl. MDR), Actinomycetes, Adenovirus, Aeromonasspp., Alcaligenes faecalis, Alcaligenes spp./Achromobacter spp.,Alcaligenes xylosoxidans (incl. ESBL/MRGN), Arbovirus, Aspergillus spp.,Astrovirus, Bacillus anthracis, Bacillus cereus, Bacillus subtilis,Bacteroides fragilis, Bartonella quintana, Bordetella pertussis,Borrelia burgdorferi, Borrelia recurrentis, Brevundimonas diminuta,Brevundimonas vesicularis, Brucella spp., Burkholderia cepacia (incl.MDR), Burkholderia mallei, Burkholderia pseudomallei, Campylobacterjejuni/coli, Candida albicans, Candida krusei, Candida parapsilosis,Chikungunya virus (CHIKV), Chlamydia pneumoniae, Chlamydia psittaci,Chlamydia trachomatis, Citrobacter spp., Clostridium botulinum,Clostridium difficile, Clostridium perfringens, Clostridium tetani,Coronavirus (incl. SARS- and MERS-CoV), Corynebacterium diphtheriae,Corynebacterium pseudotuberculosis, Corynebacterium spp.,Corynebacterium ulcerans, Coxiella burnetii, Coxsackievirus,Crimean-Congo haemorrhagic fever virus, Cryptococcus neoformans,Cryptosporidium hominis, Cryptosporidium parvum, Cyclosporacayetanensis, Cytomegalovirus (CMV), Dengue virus, Ebola virus,Echovirus, Entamoeba histolytica, Enterobacter aerogenes, Enterobactercloacae(incl. ESBL/MRGN), Enterococcus faecalis (incl. VRE),Enterococcus faecium (incl. VRE), Enterococcus hirae, Epidermophytonspp., Epstein-Barr virus (EBV), Escherichia coli (incl. EHEC, EPEC,ETEC, EIEC, EAEC, ESBL/MRGN, DAEC), Foot-and-mouth disease virus (FMDV),Francisella tularensis, Giardia lamblia, Haemophilus influenzae,Hantavirus, Helicobacter pylori, Helminths (Worms), Hepatitis A virus(HAV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis Dvirus, Hepatitis E virus, Herpes simplex virus (HSV), Histoplasmacapsulatum, Human enterovirus 71, Human herpesvirus 6 (HHV-6), Humanherpesvirus 7 (HHV-7), Human herpesvirus 8 (HHV-8), Humanimmunodeficiency virus (HIV), Human metapneumovirus, Humanpapillomavirus, Influenza virus, Klebsiella granulomatis, Klebsiellaoxytoca (incl. ESBL/MRGN), Klebsiella pneumoniae MDR (incl. ESBL/MRGN),Lassa virus, Leclercia adecarboxylata, Legionella pneumophila,Leishmania spp., Leptospira interrogans, Leuconostocpseudomesenteroides, Listeria monocytogenes, Marburg virus, Measlesvirus, Micrococcus luteus, Microsporum spp., Molluscipoxvirus,Morganella spp., Mumps virus, Mycobacterium chimaera Myco, Mycobacteriumleprae Myco, Mycobacterium tuberculosis (incl. MDR), Mycoplasmagenitalium, Mycoplasma pneumoniae, Neisseria meningitidis, Neisseriagonorrhoeae, Norovirus, Orientia tsutsugamushi, Pantoea agglomerans,Parainfluenza virus, Parvovirus, Pediculus humanus capitis, Pediculushumanus corporis, Plasmodium spp., Pneumocystis jiroveci, Poliovirus,Polyomavirus, Proteus mirabilis(incl. ESBL/MRGN), Proteus vulgaris,Providencia rettgeri, Providencia stuartii, Pseudomonas aeruginosa,Pseudomonas spp., Rabies virus, Ralstonia spp., Respiratory syncytialvirus (RSV), Rhinovirus, Rickettsia prowazekii, Rickettsia typhi,Roseomonas gilardii, Rotavirus, Rubella virus, Salmonella enteritidis,Salmonella paratyphi, Salmonella spp., Salmonella typhimurium, Sarcoptesscabiei (Itch mite), Sapovirus, Serratia marcescens (incl. ESBL/MRGN),Shigella sonnei, Sphingomonas species, Staphylococcus aureus(incl. MRSA,VRSA), Staphylococcus capitis, Staphylococcus epidermidis(incl. MRSE),Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcuslugdunensis, Staphylococcus saprophyticus, Stenotrophomonas maltophilia,Streptococcus pneumoniae, Streptococcus pyogenes (incl. PRSP),Streptococcus spp., TBE virus, Toxoplasma gondii, Treponema pallidum,Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp.,Trichosporon spp., Trypanosoma brucei gambiense, Trypanosoma bruceirhodesiense, Trypanosoma cruzi, Vaccinia virus, Varicella zoster virus,Variola virus, Vibrio cholerae, West Nile virus (WNV), Yellow fevervirus, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis, Zika virus, and the like.

In some instances, a multi-assay processing, analysis or detectionmethod may find use in simultaneous or co-timely or overlappingprocessing, analysis or detection of a plurality of clinically relevanttarget nucleic acids including but not limited to e.g., target nucleicacids derived or originating from one or more clinically relevantpathogenic bacteria such as e.g., Bacillus anthracis, Bacillus cereus,Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borreliaburgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis,Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis,Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis,Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani, Corynebacteriumdiphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichiacoli, Francisella tularensis, Haemophilus influenzae, Helicobacterpylori, Legionella pneumophila, Leptospira interrogans, Leptospirasantarosai, Leptospira weilii, Leptospira noguchii, Listeriamonocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae,Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii,Salmonella typhi, Salmonella typhimurium, Shigella sonnei,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae,Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum,Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, Yersiniapseudotuberculosis, etc.

In some instances, a multi-assay processing, analysis or detectionmethod may find use in simultaneous or co-timely or overlappingprocessing, analysis or detection of a plurality of clinically relevanttarget nucleic acids including but not limited to e.g., target nucleicacids derived or originating from one or more clinically relevantpathogenic protozoans such as e.g., protozoan parasites including butnot limited to e.g., Acanthamoeba spp., Balamuthia mandrillaris, BabesiaB. divergens, B. bigemina, B. equi, B. microfti, B. duncani, Balantidiumcoli, Blastocystis spp., Cryptosporidium spp., Cyclospora cayetanensis,Dientamoeba fragilis, Entamoeba histolytica, Giardia lamblia, Isosporabelli, Leishmania spp., Plasmodium falciparum (80% of cases), Plasmodiumvivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodiummalariae, Plasmodium knowlesi, Rhinosporidium seeberi, Sarcocystisbovihominis, Sarcocystis suihominis, Toxoplasma gondii, Trichomonasvaginalis, Trypanosoma brucei, Trypanosoma cruzi, etc.

In some instances, a multi-assay processing, analysis or detectionmethod may find use in simultaneous or co-timely or overlappingprocessing, analysis or detection of a plurality of clinically relevanttarget nucleic acids including but not limited to e.g., target nucleicacids derived or originating from one or more clinically relevantpathogenic worms such as e.g., Helminths parasites including but notlimited to e.g., Cestoda, Taenia multiceps, Diphyllobothrium latum,Echinococcus granulosus, Echinococcus multilocularis, E. vogeli, E.oligarthrus, Taenia saginata, Taenia solium, Bertiella mucronata,Bertiella studeri, Spirometra erinaceieuropaei, etc.

In some instances, a multi-assay processing, analysis or detectionmethod may find use in simultaneous or co-timely or overlappingprocessing, analysis or detection of a plurality of clinically relevanttarget nucleic acids including but not limited to e.g., target nucleicacids derived or originating from one or more clinically relevant flukesincluding but not limited to e.g., Clonorchis sinensis; Clonorchisviverrini, Dicrocoelium dendriticum, Metagonimus yokogawai, Metorchisconjunctus, Opisthorchis viverrini, Opisthorchis felineus, Clonorchissinensis, Paragonimus westermani; Paragonimus africanus; Paragonimuscaliensis; Paragonimus kellicotti; Paragonimus skrjabini; Paragonimusuterobilateralis, Schistosoma sp., Schistosoma mansoni and Schistosomaintercalatum, Schistosoma haematobium, Schistosoma japonicum,Schistosoma mekongi-, Echinostoma echinatum, Trichobilharzia regenti,Schistosomatidae, etc.

In some instances, a multi-assay processing, analysis or detectionmethod may find use in simultaneous or co-timely or overlappingprocessing, analysis or detection of a plurality of clinically relevanttarget nucleic acids including but not limited to e.g., target nucleicacids derived or originating from one or more clinically relevantroundworms including but not limited to e.g., Ancylostoma duodenale,Necator americanus, Angiostrongylus costaricensis, Ascaris sp. Ascarislumbricoides, Baylisascaris procyonis, Brugia malayi, Brugia timori,Dioctophyme renale, Dracunculus medinensis, Enterobius vermicularis,Enterobius gregorii, Halicephalobus gingivalis, Loa loa filaria,Mansonella streptocerca, Onchocerca volvulus, Strongyloides stercoralis,Thelazia californiensis, Thelazia callipaeda, Toxocara canis, Toxocaracati, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni,Trichinella nativa, Trichuris trichiura, Trichuris vulpis, Wuchereriabancrofti, etc.

In some instances, a multi-assay processing, analysis or detectionmethod may find use in simultaneous or co-timely or overlappingprocessing, analysis or detection of a plurality of clinically relevanttarget nucleic acids including but not limited to e.g., target nucleicacids derived or originating from one or more relevant other parasitesincluding but not limited to e.g., Archiacanthocephala, Moniliformismoniliformis, Linguatula serrata, Oestroidea, Calliphoridae,Sarcophagidae, Tunga penetrans, Dermatobia hominis, Acari, CimicidaeCimex lectularius, Pediculus humanus, Pediculus humanus corporis,Pthirus pubis, Demodex folliculorum/brevis/canis, Sarcoptes scabiei,Cochliomyia hominivorax, Pulex irritans, Arachnida Ixodidae andArgasidae, etc.

A multi-assay processing, analysis and/or detection method of theinstant disclosure may include any combination of assays including butnot limited to e.g., any combination of assays for detecting a targetnucleic acid derived from any combination of the organisms describedherein.

In some instances, a multi-assay processing, analysis and/or detectionmethod of the instant disclosure may include a combination of assays fordetecting two or more target nucleic acid from or derived from HIV, HCV,HBV, CT/NG (Chlamydia trachomatis (CT)/Neisseria gonorrhoeae (NG)) andHPV.

In some instances, a multi-assay processing, analysis and/or detectionmethod of the instant disclosure may include a combination of assays fordetecting two or more target nucleic acid from or derived from CMV, EBV,BK virus, MRSA, C. Diff. (Clostridium difficile) and VRE.

In some instances, a multi-assay processing, analysis and/or detectionmethod of the instant disclosure may include a combination of assays fordetecting two or more target nucleic acid from or derived fromAdenovirus, TB, VZV (Varicella-zoster virus), HSV, JC virus andEnterovirus.

In some instances, a multi-assay processing, analysis and/or detectionmethod of the instant disclosure may include a combination of assays fordetecting two or more target nucleic acid from or derived from LGV(Lymphogranuloma venereum), one or more viruses of the Respiratory ViralPanel (RVP; Human Metapneumovirus (hMPV), Rhinovirus, Influenza A,Influenza A subtype H1, Influenza A subtype H3, Influenza B, RespiratorySyncytial Virus (RSV) A, Respiratory Syncytial Virus (RSV) B,Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3,Adenovirus), HHV6 (human herpesvirus 6), Trich/Myco (Trichomonas(Trich)/Mycoplasma (Myco)) and Norovirus.

In some instances, a multi-assay processing, analysis and/or detectionmethod of the instant disclosure may include a combination of assays fordetecting two or more target nucleic acid from or derived from HIV, HCV,HBV, CT/NG, HPV, CMV, EBV, BK, MRSA, C. Diff. VRE, Adenovirus, TB, VZV,HSV, JC, Enterovirus, LGV, RVP, HHV6, Trich/Myco and Norovirus.

In some embodiments, the methods of the instant disclosure includeprocessing multiple assays according to the longest processing and/oranalysis step required for each particular assay. For example, in someinstances, a multi-assay processing method may include preparing asample processing unit (SPU) cartridge for a period of timecorresponding to the longest SPU cartridge preparation step required forall of the assays of the plurality. An SPU cartridge preparation step,as described herein, may include the aliquoting of necessary reagentsinto sample processing wells of a multi-well vessel in preparation forsample processing, e.g., lysis and extraction of nucleic acids. SPUcartridge preparation steps for different assays will vary, e.g.,because certain assays may require more or less reagents than anotherassay.

In some instances, a multi-assay processing method may include a sampleloading step that is performed for a period of time corresponding to thelongest sample loading step required for all of the assays of theplurality. A sample loading step, as described herein, may include theloading of the sample into the SPU cartridge of a particular assay.Sample loading steps may vary, e.g., because a particular assay mayrequire more or less sample than another assay.

In some instances, a multi-assay processing method may include a sampleprocessing step (e.g., as performed by a SPU module) that is performedfor a period of time corresponding to the longest sample processing steprequired for all of the assays of the plurality. Sample processing stepsinclude but are not limited to sample lysis (including the chemical,physical and/or temporal components thereof), washing steps (includingbut not limited to one or more washing steps including one washing step,two washing steps, three washing steps, etc.), nucleic acid elution,etc. The length of sample processing steps for different assays willvary for numerous reasons including but not limited to e.g., because alonger or shorter lysis time may be required for a particular organismor cell from which nucleic acid is to be extracted, because more or lesswash steps are required to sufficiently clean the extracted nucleic acidbefore amplification and detection, because elution times may vary, etc.

In some instances, a multi-assay processing method may include a nucleicacid amplification and analysis step that is performed for a period oftime corresponding to the longest nucleic acid amplification andanalysis step required for all of the assays of the plurality. Nucleicacid amplification and analysis steps as described herein will generallyrefer to but are not limited to real-time PCR amplification and analysissteps. The necessary time period required for nucleic acid amplificationand analysis for a particular assay will vary for numerous reasonsincluding but not limited to e.g., the likely starting amount of targetnucleic acid, the hybridization efficiency of the particular primers ofthe assay, the length of the amplicon, the amount of amplificationrequired for sufficient detection, etc.

The different steps of a multi-assay processing method may representatomic operations, where an atomic operation in a multi-assay processinginstrument may be allocated a fixed amount of time in a lockstepprotocol. Atomic operation length for various steps in an assay (e.g.,sample loading steps, sample processing steps, nucleic acidamplification and analysis steps, etc.) may be determined by comparingthe length of time required to complete the particular step for each ofthe various assays and identifying that which requires the longestamount of time across all assays. Accordingly, various steps of asubject lockstep protocol may be referred herein, in some instances, asatomic operations.

In some embodiments, the analysis step of the amplification and analysisstep may be standardized across assays. For example, a method ofmulti-assay analysis (e.g., quantification) may include scanning with anoptical detector at a regular interval, e.g., where the interval is setand does not vary either during the amplification or across assays. Insuch instances, the nucleic acid amplification protocol used may beconsidered to be a single protocol where the invariant characteristicsof the protocol include the scan frequency and the overall length of theamplification. However, other components of the amplification protocol(e.g., the annealing times, the ramp times, the melt times, theannealing temperature, the melt temperature, etc.) need not be fixed andmay vary from one assay to another provided common measurementtimepoints may be aligned sufficient for quantification of the nucleicacid amplification in each assay.

Common measurement timepoints may be aligned, e.g., by staggering thestart (e.g., by delaying the start of a second assay after a first assayhas begun) such that the assay protocols align with the optical scanningdevice at nearly equivalent points in the amplification reaction. Forexample, in some instances, assay starts may be staggered such that, atthe moment the optical scanning device passes, each assay is at a nearlyequivalent point in the amplification reaction. The desired nearlyequivalent point will vary and may include e.g., the end of theannealing step, the start of a ramp step, etc.

In some instances, the initiation of amplification protocols in spacedreaction vessels need not be staggered. For example, in some instances,the scan speed of a analysis unit is sufficiently fast such thatamplification protocols initiated at the same time but performed inreaction vessels some distance apart can be scanned in sufficientlyrapid succession to produce measurements that are at essentially thesame relative time point in the amplification cycle or at least closeenough time points in the amplification cycle that they are comparable.

In some instances, a multi-assay processing method may include arehydrating step that is performed for a period of time corresponding tothe longest rehydration step required for all of the assays of theplurality. Rehydration steps include but are not limited to rehydrationof lyophilized reagents (including but not limited to e.g., lyophilizedbuffer, lyophilized primers, lyophilized dNTPs, etc.). The length ofrehydration steps for different assays will vary for numerous reasonsincluding but not limited to e.g., the number of reagents to berehydrated because, for example, different assays may include e.g.,different numbers or primers and/or primer pairs, etc.

In some instances, a multi-assay processing method may include apretreating step that is performed for a period of time corresponding tothe longest pretreating step required for all of the assays of theplurality. Pretreating steps include but are not limited to contactingthe sample with a protease, e.g., contacting the sample with a proteaseprior to lysis of the sample. The length of pretreating steps fordifferent assays will vary for numerous reasons including but notlimited to e.g., the necessity of pretreatment, the particularpretreatment reagents used (e.g., the particular protease or proteasesused), etc.

In some instances, a multi-assay processing method may include anelution step that is performed for a period of time corresponding to thelongest elution step required for all of the assays of the plurality.Elution steps include but are not limited to contacting a solid support(e.g., a bead, a particle, a membrane, a filter, etc.) adhered to thenucleic acid from the lysed sample with a solution of buffer sufficientto dissolve and remove the nucleic acid from the solid support. Thelength of elution steps for different assays will vary for numerousreasons including but not limited to e.g., the amount of nucleic acidexpected to be isolated, the physical and/or chemical characteristics ofthe isolated nucleic acid expected of the sample, the elution bufferused, etc.

In some instances, a multi-assay processing method may include one ormore lysis/eluate transfer steps that are performed for period(s) oftime corresponding to the longest lysis/eluate transfer step requiredfor all of the assays of the plurality. Lysis/eluate transfer stepsinclude but are not limited to transferring the lysed sample to aseparate vessel, transferring the eluate to a separate vessel (e.g., areaction vessel) and/or any physical movement steps required by a deviceto achieve such processes. The length of lysis/eluate transfer steps fordifferent assays will vary for numerous reasons including but notlimited to e.g., the amount of lysed sample, the amount of eluate, etc.

The methods of multi-assay processing and analysis as described hereinprovide for simplified programming (e.g., software programming) of anautomated multi-assay processing/analysis device by limiting schedulingcomplexity for steps of the automated processes, including sampleprocessing and analysis. The multi-assay methods allow for theprocessing and/or analysis of multiple different assays simultaneously.As described herein, corresponding steps of different assays may beallocated the same amount of time, even in instances where thecorresponding steps do not require the same amount of time in theplurality of assays. In some instances, the corresponding steps (e.g.,bulk filling step, pipetting step, SPU cartridge preparation step,sample addition step, sample processing step, etc.) of different assaysmay be each allocated a fixed amount of time (e.g., where the fixedamount of time corresponds to the longest period of time required forthe particular step out of all the different assays).

In some instances, methods performed using devices of the instantdisclosure will eliminate resource contention that results from limitingresources of the device. A device of the instant disclosure may includea limiting resource that is utilized in more than one process of thedevice such that when parallel batches are processed the resource may,unless precautions are taken, be required for two processes (i.e., onein each parallel batch) simultaneously. Resources of the device forwhich resource contention is of issue, as described herein, generallyinclude device hardware resources such as e.g., robotic components(e.g., liquid handling (e.g., bulk filling and/or pipetting) robots,vessel (e.g., SPU cartridge and/or reaction vessel) transport robots,sample processing robots, analysis (e.g., data capture) robots, wastetransport robots, and the like). System resources that will generallynot be of issue in resource contention, as described herein, includee.g., consumable resources, such as e.g., reagents, vessels, etc.

Methods of the instant disclosure eliminate such resource contention bydeploying a common lockstep protocol that includes one or more delaypoints within or between steps of the protocol. For example, in someinstances, a method of the instant disclosure may include a commonlockstep protocol that includes a delay point within a SPU cartridgepreparation step or between a SPU cartridge preparation step and a nextstep of the protocol. Such a delay point may be appropriate where, e.g.,a resource limiting component is utilized in the SPU cartridgepreparation step and/or an adjacent step of the protocol.

In some instances, a method of the instant disclosure may include acommon lockstep protocol that includes a delay point within a sampleloading step or between a sample loading step and a next step of theprotocol. Such a delay point may be appropriate where, e.g., a resourcelimiting component is utilized in the sample loading step and/or anadjacent step of the protocol.

In some instances, a method of the instant disclosure may include acommon lockstep protocol that includes a delay point within a sampleprocessing step or between a sample processing step and a next step ofthe protocol. Such a delay point may be appropriate where, e.g., aresource limiting component is utilized in the sample processing stepand/or an adjacent step of the protocol.

In some instances, a method of the instant disclosure may include acommon lockstep protocol that includes a delay point within a nucleicacid amplification/analysis step or between a nucleic acidamplification/analysis step and a next step of the protocol. Such adelay point may be appropriate where, e.g., a resource limitingcomponent is utilized in the amplification/analysis step and/or anadjacent step of the protocol.

In some instances, a method of the instant disclosure may include acommon lockstep protocol that includes a delay point within arehydrating step or between a rehydrating step and a next step of theprotocol. Such a delay point may be appropriate where, e.g., a resourcelimiting component is utilized in the rehydrating step and/or anadjacent step of the protocol.

In some instances, a method of the instant disclosure may include acommon lockstep protocol that includes a delay point within apretreating step or between a pretreating step and a next step of theprotocol. Such a delay point may be appropriate where, e.g., a resourcelimiting component is utilized in the pretreating step and/or anadjacent step of the protocol.

In some instances, a method of the instant disclosure may include acommon lockstep protocol that includes a delay point within an elutionstep or between an elution step and a next step of the protocol. Such adelay point may be appropriate where, e.g., a resource limitingcomponent is utilized in the elution step and/or an adjacent step of theprotocol.

In some instances, a method of the instant disclosure may include acommon lockstep protocol that includes a delay point within one or morelysis/eluate transfer steps or between one or more lysis/eluate transfersteps and a next step of the protocol. Such a delay point may beappropriate where, e.g., a resource limiting component is utilized inthe one or more lysis/eluate transfer steps and/or an adjacent step ofthe protocol.

A conventional sample processing/analysis protocol not employing themethods of the present disclosure but using an automated device that issubject to resource contention is depicted in the decision tree of FIG.5. As shown, once the sample process is begun, each processing step ispreceded by a decision where the device must determine whether anecessary resource for the next step is or is not available. Forexample, prior to initiating a bulk fill step, the device must determinewhether the bulk filling robot is or is not in use. If the bulk fillingrobot is in use then the device must wait until the bulk filling robotbecomes available before proceeding to the bulk fill step. Similarly,prior to performing the sample filling step, the device must determinewhether or not the liquid handling robot is or is not in use. If theliquid handling robot is in use then the device must wait until theliquid handling robot is available before proceeding to the samplefilling step. The requirement for such decisions continues at each stepwhere a limiting resource is employed. Scheduling complexity in such asystem is amplified where many different sample processes are desiredand entry of new samples into the system is unpredictable (such as in aclinical laboratory).

In embodiments of the present methods employing a lockstep protocol,delays may be configured into the protocol at predetermined and definedpositions. For example, as depicted in FIG. 6, predetermined delays areinserted into the protocol before or between individual process steps(i.e., a delay is inserted prior to bulk filling, a delay is insertedbetween bulk filling and sample filling, a delay is inserted betweensample filling and sample processing, etc.). Although depicted before orbetween steps of the protocol in FIG. 6, such delays may also beinserted within a step. Such delays are not the result of waiting forthe availability of a limiting resource but instead specificallydesigned such that parallel sample processes do not require the samelimiting resource at a given time. Unlike waiting for availability of alimiting resource as depicted in FIG. 5, the delays of FIG. 6 are notintroduced because a resource needed for the next step is in use.Instead, the delays assure that resource contention does not occur thuseliminating unplanned waiting for the availability of a limitingresource. As such, resource availability (i.e., “in use”) decisions arenot required. Although the example depicted in FIG. 6 presents a delaybetween each step, such is not necessarily required as the number ofdelays present in a lockstep protocol of the present methods may, asdescribed above, vary in presence/absence, number, frequency and length.

Idealized lockstep methods (i.e., lockstep methods that do not takeresource contention into account or lockstep methods performed ondevices configured with no limiting resources) may be modified toinclude a delay step where the method is employed on a device withlimiting resources. Such devices include those having one or morelimiting components, including of those components described herein. Insome instances a modified lockstep method to eliminate resourcecontention may be employed on a device having e.g., one roboticpipettor, one bulk filling robot, one waste robot, one cartridgehandling robot, or a device having some combination of such limitingresources. Limiting resources are also not limited to devices havingonly one of a particular resource and may include e.g., those havingtwo, three, four, five, six, seven, eight, nine or even ten or more of aparticular resource provided the particular device is configured toprocess a sufficient number of batches to induce resource contention.

In some instances, the described method may be employed to complete,from start (i.e., initial sample aspiration/preparation) to finish(i.e., data acquisition and storage/transfer), up to 96 operations orgreater in a 8 hour period, including but not limited to e.g., 108operations or greater, 120 operations or greater, 132 operations orgreater, 144 operations or greater, 156 operations or greater, 168operations or greater, 180 operations or greater, 192 operations orgreater, 204 operations or greater, 216 operations or greater, 228operations or greater, 240 operations or greater, 252 operations orgreater, 264 operations or greater, 276 operations or greater, 288operations or greater, 300 operations or greater, etc., where by“operation” is meant an analysis and/or detection method for aparticular nucleic acid analyte run parallel with at least one otheranalysis and/or detection method for a different nucleic acid analyte(e.g., a HIV assay run in parallel with a HCV assay). Such operationthroughput may be achieved taking resource contention into account,including e.g., where the subject device includes a single roboticpipettor, a single bulk filling robot, a single waste handling robot, asingle SPU cartridge handling robot, and four amplification/analysisunits (each holding twelve reaction vessels and a single analysisrobot).

In some instances, throughput of up to 288 operations or greater per 8hour period may be achieved, taking resource contention into account,including e.g., where the subject device includes a single roboticpipettor, a single bulk filling robot, a single waste handling robot, asingle SPU cartridge handling robot, and four amplification/analysisunits (each holding twelve reaction vessels and a single analysisrobot), where an operation includes an analysis and/or detection methodfor a particular nucleic acid analyte run parallel with at least twoother analysis and/or detection methods for different nucleic acidanalytes.

Furthermore, an ordinary skilled artisan will readily understand thatthe addition of particular limiting resources to a device for which acommon lockstep protocol has been designed to eliminate resourcecontention may allow for modification of the common lockstep protocol,e.g., to decrease the cadence. For example, where a particular resourceis limiting and a common lockstep protocol is configured to eliminatecontention of the resource, when a duplicate of the limiting resource isadded to the device the common lockstep protocol may be modified, e.g.,by the removal of one or more delay steps, to shorten the cadence ascompared to the initial common lockstep protocol. Accordingly, theinstant disclosure encompasses common lockstep protocols derived bydecreasing resource limitation of a device and modifying the commonlockstep protocol, including e.g., where the modification results in acadence that is modified, e.g., decreased.

Devices and Systems

The instant disclosure provides for devices and systems, e.g., automatedmulti-assay processing/analysis devices and systems, that functionaccording to the methods as described herein. Such devices and systemswill include a plurality of modules that are coordinated, by one or morecentralized controllers, to operate the system or device according tothe methods as described herein.

In some instances, the methods as described herein find use in a systemor one or more components of a system for automated analysis and sampleanalysis systems as described in e.g., Attorney Docket No. ADDV-054WO,which claims priority to U.S. Ser. No. 62/308,617 and U.S. Ser. No.62/357,772, the disclosures of which are incorporated herein byreference in their entireties.

In some instances, a multi-assay processing/analysis system of theinstant disclosure will include a sample processing unit (SPU) cartridgepreparation module, a sample loading module, a sample processing module(i.e., a SPU module) and/or a nucleic acid amplification and analysismodule. Such systems will generally require control circuitry that isconfigured with non-transitory programing to operate components of thedevice or system to perform a method as described herein.

In some instances, the methods as described herein find use inconjunction with a SPU system or component thereof, including but notlimited to e.g., a SPU cartridge or one or more parts thereof asdescribed in e.g., Attorney Docket No. ADDV-055WO, which claims priorityto U.S. Ser. No. 62/308,618, the disclosures of which are incorporatedherein by reference in their entireties. In some instances, the methodsas described herein also find use in conjunction with a nucleic acidamplification and detection device, system and/or method or a componentthereof as described in e.g., Attorney Docket No. ADDV-058WO, whichclaims priority to U.S. Ser. No. 62/308,632, the disclosures of whichare incorporated herein by reference in their entireties.

In some instances, a multi-assay processing/analysis system of theinstant disclosure will also include a pipette module (e.g., a roboticpipettor) for performing various automated pipetting functions for oneor more modules of the device. For example, in some instances a roboticpipettor may be used for rehydrating lyophilized reagents, as part of aSPU cartridge preparation module, as part of a sample loading module,and or a combination thereof. In some instances, separate pipettemodules may be used for one or more functions of the method.

In some instances, a multi-assay processing/analysis system of theinstant disclosure will also include an SPU configured for pre-treatingeach sample prior to processing the sample, a liquid transfer moduleand/or a reaction transfer module.

Multi-assay automated systems of the present disclosure include a SPUmodule. The SPU module will generally include components necessary forthe filling of a SPU cartridge, where an SPU cartridge may be amulti-well device that contains all or nearly all of the reagentsnecessary for the processing of an assay as described herein. In otherinstances, an SPU module may rely on another component of the system,e.g., the pipette module for SPU cartridge filling. SPU modules mayfurther include components for sample processing including but notlimited to e.g., components for the pretreatment of samples, componentsfor the chemical, enzymatic and/or mechanical lysis of samples,components for the washing of samples and/or sample analytes, componentsfor the elution of nucleic acid analytes, etc.

SPU cartridges may be prepared in a SPU cartridge preparation position.The preparation may include one or more (e.g., 2 or more) SPU cartridgepreparation positions, where SPU cartridges are transported to the oneor more SPU cartridge preparation positions by a robotic SPU cartridgehandler. Depending on the particular configuration of the system, theSPU cartridges transported to the preparation positions may be empty ormay include samples (e.g., omitting the need for a sample transferstep). In some instances, a SPU cartridge may include most if not all ofthe reagents necessary for the sample preparation process (e.g.,eliminating the need for further setup steps of the SPU cartridge).

Sample loading modules of the subject disclosure include a liquidhandling robot (e.g., a robotic pipettor) configured to aspirate all ora portion of a sample and dispense it into a SPU cartridge according toinstructions received from programming. Accordingly, sample loadingmodule may be controlled by circuitry configured to control modulecomponents of a multi-assay system according to the methods describedherein.

Sample processing modules of the subject disclosure include devices forthe physical manipulations of samples required to isolate nucleic acidfrom the sample. For example, in some instances, a sample processingmodule may include a plunger for physical agitation of the sample topromote lysis. A sample processing module may also include amagnetize-able rod for use in manipulating magnetic beads or othermagnetic solid support for nucleic acid of the sample. For example, insome instances, following lysis in the sample processing module,magnetic beads or particles may be used to bind nucleic acid and themagnetic beads or particles may be extracted, carrying the nucleic acid,using a magnetize-able rod. In some instances, the plunger may serve asthe magnetize-able rod, e.g., through insertion of a magnet into theplunger. The same processing module may further include mechanisms fortransferring nucleic acid between wash wells, including e.g., where themagnetize-able rod or a magnetize-able plunger serves such a purpose. Inaddition, the sample processing module may further be configured toallow for the elution of nucleic acid from a bound solid support, suchas magnetic beads.

Accordingly, the sample processing module may perform a variety ofsample processing functions and will include the necessary componentsfor serving such functions. As such, the individual functions of thesample processing unit (i.e., physical manipulations, lysis, elution,etc.) may be coordinated into a multi-assay protocol as described hereinwhere, e.g., the length of any one particular step may be increased fora particular assay to match the time required for the step for the assayin which the particular step takes the longest. In some instances, onlythe overall length of the sample processing step will be coordinated ina multi-assay protocol including e.g., the sub-steps of sampleprocessing (i.e., physical manipulations, lysis, elution, etc.) may notbe coordinated.

In some instances, the methods as described herein may be applied to orused in conjunction with a sample processing device and/or a sampleprocessing method as described in e.g., Attorney Docket No. ADDV-059WO,which claims priority to U.S. Ser. No. 62/308,645, the disclosures ofwhich are incorporated herein by reference in their entireties.

Nucleic acid amplification and analysis modules of the instantdisclosure will generally include the components of a thermocycler andan optical detection system. Where electricity is employed to controlthermal cycling, at a minimum, a thermocycler useful in nucleic acidamplification with include a thermal block, a thermoelectric cooler anda control unit, such components configured together to regulate thetemperature of a reaction vessel in a controlled manner so as to cyclethe reaction through multiple rounds of heating and cooling through adefined series of temperature steps. A nucleic acid amplification deviceof the instant disclosure may include thermoregulatory components inaddition to the thermal block and thermoelectric cooler including butnot limited to e.g., a heatsink, a fan, a duct, a vent, etc. Two or morethermoregulatory components of a nucleic acid amplification device willgenerally be in thermal contact with one another.

The analysis component of a nucleic acid amplification and analysismodule will generally include a multi-reaction analysis devicesconfigured for the analysis of multiple amplification reaction vesselsduring the amplification reactions. Multi-reaction analysis devices ofthe instant disclosure allow for the monitoring of multiple real-timePCR reactions. Such multi-reaction analysis devices include opticalcomponents, conveyor components and signal detection/processingcomponents wherein such components are configured for the frequentmonitoring of multiple reaction vessels.

Multi-reaction analysis devices of the instant disclosure includeoptical components sufficient for the optical analysis of nucleic acidamplification reactions, including real-time PCR reactions, as describedherein. Such optical components will include illumination components,including one or more excitation components, and components forreceiving emission light from the reaction vessel. In certainembodiments a linear conveyer is paired with linearly arranged opticalcomponents and linearly arranged reaction vessels to allow for thescanning of the optical components, by means of the conveyor, past thereaction vessels to mediate the analysis. As such, in some instances,control circuitry is configured to regulate the rate and/or interval ofscanning of the optical detector to operate the system according to themethods as described herein. In some instances, the scanning internal isinvariant and the control circuitry maintains a constant rate and/orinterval of scanning. In other instances, the scanning interval isvariant.

Systems of the instant disclosure may include various robotic handlingcomponents, including but not limited to e.g., a robotic SPU cartridgehandler, a liquid handling robot, a bulk filling robot, a waste robot,and the like. Such robotic components may function to distribute SPUcartridges to various locations throughout the system including but notlimited to e.g., a bulk filling station, a pipetting station, a samplefilling station, a sample processing station, a waste station, etc.,according to instructions received from programming. In some instances,systems of the instant disclosure may include a liquid handling robotwhere such a robot may contain an automated pipetting system fordispensing and/or aspirating liquids according to instructions receivedfrom programming. In some instances, a control circuit of the instantdisclosure may include programming configured to control the robotichandling components according to the methods described herein. In someinstances, a liquid transfer module may include a liquid handing robotconfigured to dispense and/or aspirate liquid according to instructionsreceived from programming.

Robotic handlers of the instant disclosure are not limited to thoseconfigured to relocate SPU cartridges and liquids and may also includee.g., a reaction transfer module configured to transfer a reactionvessel to another location, e.g., to transfer from a sample preparationand/or processing location to an amplification/detection location,according to instructions received from programming. In some instances,components of a liquid handling robot may serve to handle non-liquidcomponents including but not limited to serving as a reaction vesseltransfer module.

As described herein, the various components of the multi-assayprocessing/analysis system may be configured according to a method witha plurality of steps which, although different in length as theyproceed, are made the same length across all assays and may includedelay periods within and/or between steps to function as a commonlockstep protocol for all assays. Such coordinated processing andanalysis is made possible by hardware and software programing of controlcircuitry configured to operate the various system components accordingto the unified protocol. As such, the advance from one component toanother may be pre-timed. However, in certain instances, steps and/orprocesses may require input, execution or other trigger to proceed and,as such, components of the system may be in electrical communicationwith one another.

In some instances, the components of the systems as described herein maybe connected by a wired data connection. Any suitable and appropriatewired data connection may find use in connecting the components of thedescribed systems, e.g., as described herein, including but not limitedto e.g., commercially available cables such as a USB cable, a coaxialcable, a serial cable, a C2G or Cat2 cable, a Cat5/Cat5e/Cat6/Cat6acable, a Token Ring Cable (Cat4), a VGA cable, a HDMI cable, a RCAcable, an optical fiber cable, and the like. In some instances, e.g.,where data security is less of a concern, wireless data connections maybe employed including but not limited to e.g., radio frequencyconnections (e.g., PAN/LAN/MAN/WAN wireless networking, UHF radioconnections, etc.), an infrared data transmission connection, wirelessoptical data connections, and the like.

In certain instances, programing as described herein of the systems ofthe instant disclosure may be stored in a “memory” and/or on computerreadable memory. As such, the devices and systems of the instantdisclosure may further include a memory that is capable of storinginformation such that it is accessible and retrievable at a later dateby a computer. Any convenient data storage structure may be chosen,based on the means used to access the stored information. In certainaspects, the information may be stored in a “permanent memory” (i.e.memory that is not erased by termination of the electrical supply to acomputer or processor) or “non-permanent memory”. Computer hard-drive,CD-ROM, floppy disk, portable flash drive and DVD are all examples ofpermanent memory. Random Access Memory (RAM) is an example ofnon-permanent memory. A file in permanent memory may be editable andre-writable.

Substantially any circuitry can be configured to a functionalarrangement within the devices and systems for performing the methodsdisclosed herein provided the described considerations are followed.However, as described herein, systems employing the instant methods willgenerally make use of hardware configurations compatible with thedisclosed unified processing and analysis protocols.

The hardware architecture of such circuitry, including e.g., aspecifically configured computer, is well known by a person skilled inthe art, and can comprise hardware components including one or moreprocessors (CPU), a random-access memory (RAM), a read-only memory(ROM), an internal or external data storage medium (e.g., hard diskdrive). Such circuitry can also comprise one or more graphic boards forprocessing and outputting graphical information to display means. Theabove components can be suitably interconnected via a bus within thecircuitry, e.g., inside a specific-use computer. The circuitry canfurther comprise suitable interfaces for communicating withgeneral-purpose external components such as a monitor, keyboard, mouse,network, etc. In some embodiments, the circuitry can be capable ofparallel processing or can be part of a network configured for parallelor distributive computing to increase the processing power for thepresent methods and programs. In some embodiments, the program code readout from the storage medium can be written into a memory provided in anexpanded board inserted in the circuitry, or an expanded unit connectedto the circuitry, and a CPU or the like provided in the expanded boardor expanded unit can actually perform a part or all of the operationsaccording to the instructions of the programming, so as to accomplishthe functions described.

In addition to the components of the devices and systems of the instantdisclosure, e.g., as described above, systems of the disclosure mayinclude a number of additional components, such as data output devices,e.g., monitors and/or speakers, data input devices, e.g., interfaceports, keyboards, etc., actuatable components, power sources, etc.

Computer Readable Media

The instant disclosure includes computer readable medium, includingnon-transitory computer readable medium, which stores instructions formethods described herein. Aspects of the instant disclosure includecomputer readable medium storing instructions that, when executed by acomputing device, cause the computing device to perform one or moresteps of a method as described herein.

In certain embodiments, instructions in accordance with the methodsdescribed herein can be coded onto a computer-readable medium in theform of “programming”, where the term “computer readable medium” as usedherein refers to any storage or transmission medium that participates inproviding instructions and/or data to a computer for execution and/orprocessing. Examples of storage media include a floppy disk, hard disk,optical disk, magneto-optical disk, CD-ROM, CD-R, magnetic tape,non-volatile memory card, ROM, DVD-ROM, Blue-ray disk, solid state disk,and network attached storage (NAS), whether or not such devices areinternal or external to the computer. A file containing information canbe “stored” on computer readable medium, where “storing” means recordinginformation such that it is accessible and retrievable at a later dateby a computer.

The computer-implemented method described herein can be executed usingprogramming that can be written in one or more of any number of computerprogramming languages. Such languages include, for example, Java (SunMicrosystems, Inc., Santa Clara, Calif.), Visual Basic (Microsoft Corp.,Redmond, Wash.), and C++ (AT&T Corp., Bedminster, N.J.), as well as anymany others.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1: Generation of a Common Lockstep Sample ProcessingProtocol

The instant example describes the creation of a single lockstep assaytiming protocol that harmonizes the processing for each type of assayinto a common assay timing protocol where all assays result in the sametime and throughput regardless of which assay or mix of assays are beingrun on the automated device.

The pipetting step, SPU cartridge setup step, sample addition step,pretreatment step, digestion transfer step, SPU processing step, eluatetransfer step and amplification/detection step was determined forvarious assays including HIV, HCV, CT/NG and HBV. The longest of eachstep (i.e., the longest pipetting step, the longest SPU cartridge setupstep, the longest sample addition step, the longest pretreatment step,the longest digestion transfer step, the longest SPU processing step,the longest eluate transfer step and the longest amplification/detectionstep) for each assay were compiled into a single common idealized“lockstep” protocol. FIG. 1 provides an example of how an idealizedcommon lockstep assay timing protocol was derived from several assays(e.g., HIV, HCV, CT/NG and HBV) that have unique processing steps andtimes.

The idealized lockstep timing protocol provided in FIG. 1 does not takeinto account the limited resources of an actual device (e.g., a deviceconfigured to have a single robotic pipettor, a single bulk fillingrobot, a single waste handling robot, a single SPU cartridge handlingrobot, etc.). To generate sequences which allow operation on a number ofbatches within a system simultaneously using limited resources, theidealized lockstep protocol of FIG. 1 was used as a starting point andmodified to eliminate resource contention. For example, FIG. 2illustrates how four different assay types (HIV, HCV, CT/NG and HBV),utilizing an idealized common lockstep assay timing protocol would beprocessed in an idealized system without considering for resourcecontention (i.e., if a dedicated resource for all processing stepslisted in the table in the figure were present for each batch). However,if SPU setup and sample addition were to use the same resource (e.g., asingle robotic pipettor) and/or sample processing and pretreatment wereto use the sample resource (e.g., a single robotic pipettor), resourcecontention would occur (as indicated on FIG. 2 as vertical arrows) andmuch greater staggering of processing steps would be required.

Rather than simply restricting batch processing to a serial protocolwhere resource contention is alleviated by preventing the initiation ofa new batch until a previous batch is complete, the lockstep protocolwas modified to include a sequence of delays inserted between and/orwithin steps to generate a modified common lockstep protocol having anoptimized cadence (i.e., batch per unit time) taking into account allpotential assays to be run on the device.

For example, as depicted in FIG. 3, when resources are unlimited(“Unlimited Resource Case”) a lockstep protocol may concurrently processsuccessively initiated batches without concern for resource contention.However, when resources are limiting (“Limited Resources with ResourceContention”), e.g., where only three resources are available thatperform “step 2”, resource contention (as indicated with underlining)occurs between e.g., the third and fourth batches at the initiation of“step two” of the fourth batch because the first batch has yet tocomplete “step 2” and all three of the available resources are occupied.In the “Limited Resources with Resource Contention” example, furtherresource contention (also indicated in FIG. 3 with underlining) is seenwhen assuming that only one resource is available that performs each of“step 1”, “step 3” and “step 5”.

These resource contentions were eliminated (as shown in the “ContentionsEliminated” panel of FIG. 3) by modeling the actual processing of aresource limited device to identify resource contentions and determinewhere the addition of delay points (“Delay”) would prevent such resourcecontention and produce an optimized cadence.

This common lockstep protocol with added delay points allows for theconcurrent (simultaneous and/or overlapping) processing/analysis ofdifferent assays without affecting throughput and eliminating resourcecontention. Furthermore, the lockstep protocol allows initiation of anadditional different assay or a new batch of an already running assayduring the processing of a previously started assay without affectingthe processing of either assay.

The lockstep protocol further simplifies automated device programming(e.g., software) and operation. However, the hardware functioning in anautomated device operating under a lockstep protocol required additionaldesign considerations.

In one tested embodiment, the hardware allowed for 12 different assays(each in batches of 4 samples each) to be run at any one time on asingle device without resource contention even where resources arelimiting (e.g., where the device has a single robotic pipettor, a singlebulk filling robot, a single waste handling robot and a single SPUcartridge handling robot). Batch sizes of 4 samples allows for differentassay attributes per grouping of 4 samples, as long as the overallprocessing time is the same for all batches (i.e., all batches conformto the overall common lockstep protocol length). For example, in samplepreparation, each group of 4 samples can have a different temperaturecontrol but this will not impact the overall processing time.

The described example of a common lockstep assay timing protocolsimplified the software scheduling complexity for an automatedinstrument that processes multiple different assays simultaneously(i.e., in parallel) since each processing step/resource in the system(i.e. Pipetting, SPU Cartridge Setup, Sample Addition, etc.) wasallocated a fixed amount that included the time necessary to completethe step in the most resource intensive assay and additional added delayto align the step into an optimized cadence that eliminates contentionfor shared resources.

Furthermore, for the amplification and detection subsystem in thisembodiment, independent thermal control was provided for each set of twosamples. The system used this advanced control to delay the protocolstart for each subsequent pair of samples in order to read all samplesat common measurement time points (e.g., as close to the same relativetime in each protocol as possible).

The protocols were structured such that the amplification and detectionfor each assay fit within a common optical protocol (i.e., scanningprotocol) such that the scan stage can reach each assay at the correcttime to make a measurement. In the described example, the scan stagecontinuously scans all assays (with the option for pauses or calibrationsteps between scans) and particular data for each assay was capturedwhen relevant. FIG. 4 provides an example for such coordinated scanningin three different assays (each with a pair of reaction vessels). Thearrows and steps indicate when each assay requires a scan to occur. Ascan be seen, all three assays do not always align and only the relevantdata for each assay need be written to the output file and/or used infurther measurement and analysis.

The common lockstep operation significantly simplified the softwarescheduling complexity for the tested instrument overall. By having acommon lockstep protocol, the software followed a deterministic model ofwhen different activities needed to occur for different resources. Inthe event that a step completes early, the system is programed to waituntil the period was over to proceed forward with that step. Thisstructured scheduling ensured that there are not resource contentions.

Notwithstanding the appended claims, the disclosure is also defined bythe following clauses:

1. A method of multi-assay processing, the method comprising:

a) preparing a sample processing unit (SPU) cartridge for each of two ormore different target nucleic acid detection assays;

b) loading a sample into each prepared SPU cartridge;

c) processing each loaded SPU cartridge to isolate a sample nucleic acidfor each of the two or more different target nucleic acid detectionassays; and

d) amplifying and analyzing each sample nucleic acid for a targetnucleic acid specific to each of the two or more different targetnucleic acid detection assays, wherein the method comprises at least onedelay step within or between steps a) through d) and steps a) through d)are each performed for a time period that is equal for the two or moredifferent target nucleic acid detection assays.

2. The method according to clause 1, wherein the method comprises adelay step between steps a) and b).

3. The method according to any one of clauses 1-2, wherein the methodcomprises a delay step between steps b) and c).

4. The method according to any one of clauses 1-3, wherein the methodcomprises a delay step between steps c) and d).

5. The method according to any one of clauses 1-4, wherein the methodfurther comprises rehydrating lyophilized reagents for each of the twoor more different target nucleic acid detection assays prior to thepreparing, wherein the rehydrating is performed for a time period thatis equal for the two or more different target nucleic acid detectionassays.

6. The method according to clause 5, wherein the method comprises adelay step following the rehydrating.

7. The method according to any one of clauses 1-6, wherein the methodfurther comprises pre-treating each loaded SPU cartridge prior to theprocessing, wherein the pre-treating is performed for a time period thatis equal for the two or more different target nucleic acid detectionassays.

8. The method according to clause 7, wherein the method comprises adelay step following the pre-treating.

9. The method according to clause 7, wherein the pre-treating comprisescontacting the sample with a protease.

10. The method according to any one of clauses 1-9, wherein theprocessing comprises transferring the sample into a solution comprisinga lysis buffer, wherein the transferring is performed for a time periodthat is equal for the two or more different target nucleic aciddetection assays.

11. The method according to clause 10, wherein the method comprises adelay step following the transferring.

12. The method according to any one of clauses 1-11, wherein theprocessing comprises eluting the nucleic acid and transferring theeluted nucleic acid into a reaction vessel for the amplifying andanalyzing, wherein the eluting is performed for a time period that isequal for the two or more different target nucleic acid detectionassays.

13. The method according to clause 12, wherein the method comprises adelay step following the eluting.

14. The method according to any one of clauses 1-13, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a human immunodeficiency virus (HIV) nucleic acid.

15. The method according to any one of clauses 1-14, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a hepatitis C virus (HCV) nucleic acid.

16. The method according to any one of clauses 1-15, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a hepatitis B virus (HBV) nucleic acid.

17. The method according to any one of clauses 1-16, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Chlamydia trachomatis (CT) nucleic acid, a Neisseriagonorrhoeae (NG) nucleic acid or a combination there of.

18. The method according to any one of clauses 1-17, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Human papillomavirus (HPV) nucleic acid.

19. The method according to any one of clauses 1-18, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Cytomegalovirus (CMV) nucleic acid.

20. The method according to any one of clauses 1-19, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect an Epstein-Barr virus (EBV) nucleic acid.

21. The method according to any one of clauses 1-20, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a BK virus nucleic acid.

22. The method according to any one of clauses 1-21, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Methicillin-resistant Staphylococcus aureus (MRSA) nucleicacid.

23. The method according to any one of clauses 1-22, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Clostridium difficile (D. Diff.) nucleic acid.

24. The method according to any one of clauses 1-23, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Vancomycin-resistant Enterococcus (VRE) nucleic acid.

25. The method according to any one of clauses 1-24, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect an Adenovirus nucleic acid.

26. The method according to any one of clauses 1-25, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a tuberculosis (TB) nucleic acid.

27. The method according to any one of clauses 1-26, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Varicella-zoster virus (VZV) nucleic acid.

28. The method according to any one of clauses 1-27, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Herpes simplex virus (HSV) nucleic acid.

29. The method according to any one of clauses 1-28, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a JC virus nucleic acid.

30. The method according to any one of clauses 1-29, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect an Enterovirus nucleic acid.

31. The method according to any one of clauses 1-30, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Lymphogranuloma venereum (LGV) nucleic acid.

32. The method according to any one of clauses 1-31, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Respiratory Viral Panel (RVP) nucleic acid.

33. The method according to any one of clauses 1-32, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a human herpesvirus 6 (HHV6) nucleic acid.

34. The method according to any one of clauses 1-33, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Trichomonas (Trich) nucleic acid, a Mycoplasma (Myco)nucleic acid or a combination thereof.

35. The method according to any one of clauses 1-34, wherein the two ormore different target nucleic acid detection assays comprises an assayto detect a Norovirus nucleic acid.

36. The method according to any one of clauses 1-35, wherein the methodprocess 3 or more different target nucleic acid detection assays.

37. The method according to clause 36, wherein the method process 10 ormore different target nucleic acid detection assays.

38. A method of multi-assay quantification, the method comprising:

a) initiating a nucleic acid amplification protocol in a first samplepair;

b) scanning the first sample pair with an optical detector at a regularinterval during the nucleic acid amplification protocol, wherein theinterval allows for the collection of data by the optical detector attimepoints of the amplification protocol sufficient for quantificationof the nucleic acid amplification in the first sample pair;

c) initiating the nucleic acid amplification protocol in a second samplepair at a time that allows the second sample pair to be scanned by theoptical detector at the regular intervals and collection of data by theoptical detector at timepoints of the amplification protocol sufficientfor quantification of nucleic acid amplification in the second samplepair.

39. The method according to clause 38, wherein the initiating of thenucleic acid amplification protocol of the first sample pair and theinitiating of the nucleic acid amplification protocol of the secondsample pair occur at essentially the same time.

40. The method according to clause 38, wherein the initiating of thenucleic acid amplification protocol of the first sample pair and theinitiating of the nucleic acid amplification protocol of the secondsample pair occur at different times.

41. The method according to any one of clauses 38-40, wherein thescanning is performed three or more times during the nucleic acidamplification protocol.

42. The method according to any one of clauses 38-41, wherein theinterval allows for the collection of data by the optical detector atmore timepoints of the amplification protocol than necessary forquantification of the nucleic acid amplification in the first and secondsample pairs.

43. The method according to any one of clauses 38-42, wherein the methodfurther comprises initiating the nucleic acid amplification protocol ina third sample pair at a time that allows the third pair to be scannedby the optical detector at the regular intervals and collection of databy the optical detector at timepoints of the amplification protocolsufficient for quantification of nucleic acid amplification in the thirdsample pair.

44. The method according to clause 43, wherein the initiating of thenucleic acid amplification protocol of the first, second and thirdsample pairs occur at essentially the same time.

45. The method according to clause 43, wherein the initiating of thenucleic acid amplification protocol of the first, second and thirdsample pairs occur at different times.

46. The method according to any one of clauses 38-45, wherein the methodfurther comprises initiating the nucleic acid amplification protocol ina fourth sample pair at a time that allows the fourth pair to be scannedby the optical detector at the regular intervals and collection of databy the optical detector at timepoints of the amplification protocolsufficient for quantification of nucleic acid amplification in thefourth sample pair.

47. The method according to clause 46, wherein the initiating of thenucleic acid amplification protocol of the first, second, third andfourth sample pairs occur at essentially the same time.

48. The method according to clause 46, wherein the initiating of thenucleic acid amplification protocol of the first, second, third andfourth sample pairs occur at different times.

49. The method according to any one of clauses 38-48, wherein the methodfurther comprises initiating the nucleic acid amplification protocol ina fifth sample pair at a time that allows the fifth pair to be scannedby the optical detector at the regular intervals and collection of databy the optical detector at timepoints of the amplification protocolsufficient for quantification of nucleic acid amplification in the fifthsample pair.

50. The method according to clause 49, wherein the initiating of thenucleic acid amplification protocol of the first, second, third, fourthand fifth sample pairs occur at essentially the same time.

51. The method according to clause 49, wherein the initiating of thenucleic acid amplification protocol of the first, second, third, fourthand fifth sample pairs occur at different times.

52. The method according to any one of clauses 38-51, wherein the methodfurther comprises initiating the nucleic acid amplification protocol ina sixth sample pair at a time that allows the sixth pair to be scannedby the optical detector at the regular intervals and collection of databy the optical detector at timepoints of the amplification protocolsufficient for quantification of nucleic acid amplification in the sixthsample pair.

53. The method according to clause 52, wherein the initiating of thenucleic acid amplification protocol of the first, second, third, fourth,fifth and sixth sample pairs occur at essentially the same time.

54. The method according to clause 52, wherein the initiating of thenucleic acid amplification protocol of the first, second, third, fourth,fifth and sixth sample pairs occur at different times.

55. A multi-assay processing system, the system comprising:

a) a sample processing unit (SPU) cartridge preparation module;

b) a sample loading module;

c) a SPU processing module;

d) a nucleic acid amplification and analysis module; and

e) control circuitry configured to perform the method according to anyone of clauses 1-54.

56. The system according to clauses 55, wherein the system furthercomprises a module for rehydrating lyophilized reagents.

57. The system according to any one of clauses 55-56, wherein the SPUprocessing module is further configured for pre-treating each sampleprior to processing the sample.

58. The system according to any one of clauses 55-57, wherein the systemfurther comprises a reaction transfer module.

59. The system according to any one of clauses 55-58, wherein the systemcomprises a single robotic pipette resource that functions in the SPUcartridge preparation module.

60. The system according to clause 59, wherein the single roboticpipette resource also functions in the sample loading module.

61. The system according to any one of clauses 59-60, wherein the singlerobotic pipette resource also functions in the module for rehydratinglyophilized reagents.

62. The system according to any one of clauses 59-61, wherein the singlerobotic pipette resource also functions in the reaction transfer module.

63. The system according to any one of clauses 55-62, wherein the systemfurther comprises one or more bulk filling robots.

64. The system according to clause 63, wherein the system comprises asingle bulk filling robot.

65. The system according to any one of clauses 55-64, wherein the systemfurther comprises one or more waste robots.

66. The system according to clause 65, wherein the system comprises asingle waste robot.

67. The system according to any one of clauses 55-66, wherein the systemfurther comprises one or more SPU cartridge handling robots.

68. The system according to clause 65, wherein the system comprises asingle SPU cartridge handling robot.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1-68. (canceled)
 69. A method for performing two or more assays on twoor more samples, the method comprising: a) loading the two or moresamples into a multi-assay processing system for parallel processing ofthe two or more assays on the two or more samples, the systemcomprising: a multi-assay processor module, a processor, and anon-transitory computer readable medium programmed with instructionsthat, when executed by the processor, cause the multi-assay processingsystem to: i) analyze the time schedule for two or more assays that havetwo or more steps that utilize two or more resources of the multi-assayprocessing module, wherein the two or more assays have different timedurations for one or more of the two or more steps; ii) determine,before the initiation of the multi-assay processing, introduction of atleast one delay step within or between the two or more steps of the twoor more assays such that simultaneous processing of the two or moreassays does not require, at a given time, the same limiting resourcefrom the multi-assay processing module; and iii) introduce the at leastone delay step within or between the two or more steps of the two ormore assays in a manner that eliminates resource contention between thetwo or more resources of the multi-assay processing module therebyallowing parallel processing of the two or more assays in themulti-assay processing module; and b) initiating the multi-assayprocessing of the two or more samples in the multi-assay processingsystem.
 70. The method of claim 69, wherein the non-transitory computerreadable medium is programmed with instructions that, when executed bythe processor, cause the multi-assay processing module to allow parallelprocessing of three or more assays that have three or more steps thatutilize three or more resources of the multi-assay processing module.71. The method of claim 69, wherein each of the two or more assays isnucleic acid detection assay.
 72. The method of claim 71, wherein eachof the two or more nucleic acid detection assays comprise two or moresteps selected from: a) preparing a nucleic acid sample, b) loading theprepared nucleic acid sample, c) amplifying and analyzing the nucleicacid sample.
 73. The method of claim 72, wherein the non-transitorycomputer readable medium is programmed with instructions that, whenexecuted by the processor, cause the multi-assay processing system tointroduce the at least one delay step within or between i) steps a) andb); ii) steps b) and c); iii) or a combination of i) and iii), in amanner that eliminates resource contention between the three or moreresources of the multi-assay processing module thereby allowing parallelprocessing of the three or more assays in the multi-assay processingmodule.
 74. The method of claim 71, wherein one of the two or moredifferent target nucleic acid detection assays is an assay to detect ahuman immunodeficiency virus (HIV) nucleic acid.
 75. The method of claim71, wherein one of the two or more different target nucleic aciddetection assays is an assay to detect a human hepatitis C virus (HCV)nucleic acid.
 76. The method of claim 71, wherein one of the two or moredifferent target nucleic acid detection assays is an assay to detect ahuman hepatitis B virus (HBV) nucleic acid.
 77. The method of claim 71,wherein one of the two or more different target nucleic acid detectionassays detects a human papillomavirus (HPV) nucleic acid.
 78. The methodof claim 71, wherein one of the two or more different target nucleicacid detection assays is an assay to detect a Cytomegalovirus (CMV)nucleic acid.
 79. The method of claim 71, wherein one of the two or moredifferent target nucleic acid detection assays is an assay to detect anEpstein-Barr virus (EBV) nucleic acid.
 80. The method of claim 71,wherein one of the two or more different target nucleic acid detectionis an assay to detect detects a BK nucleic acid.
 81. The method of claim71, wherein one of the two or more different target nucleic aciddetection assays is an assay to detect a Methicillin-resistantStaphylococcus aureus (MRSA) nucleic acid.
 82. The method of claim 71,wherein one of the two or more different target nucleic acid detectionassays is an assay to detect a Clostridium difficile nucleic acid. 83.The method of claim 71, wherein one of the two or more different targetnucleic acid detection assays is an assay to detect aVancomycin-resistant Enterococcus (VRE) nucleic acid.
 84. The method ofclaim 71, wherein one of the two or more different target nucleic aciddetection assays is an assay to detect an Adenovirus nucleic acid. 85.The method of claim 71, wherein one of the two or more different targetnucleic acid detection assays is an assay to detect a tuberculosis (TB)nucleic acid.
 86. The method of claim 71, wherein one of the two or moredifferent target nucleic acid detection assays is an assay to detect aVaricella-zoster virus (VZV) nucleic acid.
 87. The method of claim 71,wherein one of the two or more different target nucleic acid detectionassays is an assay to detect a Herpes simplex virus (HSV) nucleic acid.88. The method of claim 71, wherein one of the two or more differenttarget nucleic acid detection assays is an assay to detect a JC virusnucleic acid.
 89. The method of claim 71, wherein one of the two or moredifferent target nucleic acid detection assays is an assay to detect anEnterovirus nucleic acid.
 90. The method of claim 71, wherein one of thetwo or more different target nucleic acid detection assays is an assayto detect a Lymphogranuloma venereum (LGV) nucleic acid.
 91. The methodof claim 71, wherein one of the two or more different target nucleicacid detection assays is an assay to detect a Respiratory Viral Panel(RVP) nucleic acid.
 92. The method of claim 71, wherein one of the twoor more different target nucleic acid detection assays detects a humanherpesvirus 6 (HHV6) nucleic acid.
 93. The method of claim 71, the twoor more different target nucleic acid detection assays comprises anassay to a Trichomonas (Trich) nucleic acid, an assay to detectMycoplasma (Myco) nucleic acid, or an assay to detect the combination ofTrich nucleic acid and Myco nucleic acid.
 94. The method of claim 71,wherein the two or more different target nucleic acid detection assayscomprises an assay to detect a Chlamydia trachomatis (CT) nucleic acid,an assay to detect a Neisseria gonorrhoeae (NG) nucleic acid, or anassay to detect a combination there of CT nucleic acid and NG nucleicacid.
 95. The method of claim 71, wherein the non-transitory computerreadable medium is programmed with instructions that, when executed bythe processor, cause the multi-assay processing module to allow parallelprocessing of ten or more assays that have three or more steps thatutilize three or more resources in the multi-assay processing module.96. The method of claim 71, comprising, initiating essentially at thesame time, the multi-assay processing of the two or more samples in themulti-assay processing system.